Elucidation of Reaction Mechanisms - Isotopic Labeling

Isotopic Labeling - Examples

The application of isotopic labeling experiments to the elucidation of reaction mechanisms is illustrated by the following two examples.

First of all, an example from the fundamentals of organic chemistry, namely the acid-catalysed esterification, is analysed. Subsequently, the discussion of an example from the field of biochemistry and chemistry of natural products is presented.

18O labeling for the investigation of the acid-catalysed esterification

The elucidation of the mechanism of the acid-catalysed esterification introduces the question of which starting product (carbon acid or alcohol) the oxygen atom of the product water molecule comes from or which oxygen atom is in the ester. This question can be answered by an isotopic labeling experiment in which the alcohol is labelled with the oxygen isotope 18O.

Fig.1

Esterification with an 18O-labelled alcohol.

If the reaction is carried out with an 18O-labelled alcohol, in the end of the reaction, the 18O isotope is exclusively found in the ester and not in the water. This is indicative of the fact that a nucleophilic attack of the alcohol's oxygen atom on the carboxylic acid's (green frame) carbonyl carbon has occured and that the alcoholic proton has subsequently been cleft off during the mechanism.

Fig.2

Complete mechanism of the acid-catalysed esterification.

In order to further clarify other aspects of the mechanism, such as, for example, the role of the acid catalysis, additional experiments have been necessary. These experiments are partially depicted in the section on the investigation of kinetics in the elucidation of reaction mechanisms.

Now, take a look at the practical realization of an esterification in the following two movies:

Movie (I) on the esterification of acetic acid with ethanol

Movie (II) on the synthesis of ethyl acetate

Investigation of the biosynthesis of betaenone B with 13C-labelled substrates

It has been assumed that the secondary metabolite betaenone B [1], which was isolated from the fungus Phoma betae Fr., is a so-called polyketide. Polyketides are a widespread group of secondary metabolites that all come from the same biosynthetic pathway. The basic framework of all polyketides is made up of acetate units (from acetyl CoA). Methyl branchings may arise from the incorporation of propionate instead of acetate. Likewise, these branchings may also be the result of a subsequent transfer of a methyl group from S-adenosyl methionine.

At this point, the polyketide biosynthesis does not need to be explained in more detail. Nevertheless, how could it be proven that betaenone B is a polyketide?

For this purpose, the fungus Phoma betae Fr. was cultivated in different nutrient media, which either contained the 13C-labelled substrate [1-13C] acetate, [2-13C] acetate or [Me-13C] methionine. Subsequently, each betaenone B, synthesized in one of the nutrient media, was individually investigated by 13C NMR spectroscopy. [2,3]

Bold bond: initial acetate whose C1 is labelled by a colon. Star: Methyl group that comes from L-methionine.

Fig.4

Interactive 3D molecular model: the C1 of the initial acetate units are green and the methyl groups that come from L-methionine are blue coloured.

By the amplification of the corresponding 13C NMR signals, it is shown that betaenone B is actually made up of the 13C-labelled acetate units. In addition, it has been proven that the methyl branchings do not arise from the incorporation of propionate, but that they are a result of the methyl transfer from S-adenosyl methionine, as the corresponding 13C NMR signals of the methyl groups were amplificated after the cultivation in the nutrient medium containing [Me-13C] methionine.